A 20,000 dalton cationic polypeptide has been purified to homogeneity from extracts of neonatal rat skeletal muscle. The addition of the purified peptide to dissociated cultures of embryonic rat spinal cord neurons enhances the development of choline acetyltransferase and acetylcholine synthesis activities, but does not affect the overall metabolic activity of spinal cord cultures. This polypeptide represents the only skeletal muscle- derived neurotrophic factor purified to homogeneity, and besides NGF, it is the only other neurotrophic factor which affects cholinergic development of CNS-derived neurons. Treatment of chick embryos in ovo with the partially purified peptide during the normal cell death period rescues motor neurons which otherwise would have died. Thus, this neurotrophic peptide appears to be important for the survival and development of motor neurons. Our present efforts are directed at further characterization of the effects of this factor on the in vitro and in vivo development of motor neurons by direct application and through the development of specific antibodies to the purified factor.